Benzamidine Derivatives for Treatment and Prevention of Cancer Therapy Induced Mucositis

ABSTRACT

Mucositis is the result of a complex process of interactive biologic phenomena that take place in both the epitelium and the submucosa, often leading to severe pain and increased risk of dangerous syste f48 mic infections. Mucositis is often a side effect during chemotherapy and radiation therapy. The benzamidine derivatives herein described are particularly effective for treating and preventing mucositis since they are acting simultaneously at the several phases that characterize this disease. Data supplied from the esp@cenet database

BACKGROUND OF THE INVENTION

Although significant advances have been made in the management ofpatients undergoing cancer chemotherapy and radiotherapy, manydebilitating gastrointestinal side effects remain critical issues thathave an impact on the patient management. In addition to vomiting,nausea and diarrhea, a clinically relevant adverse event is representedby mucositis. Mucositis is the result of a complex process ofinteractive biologic phenomena that take place in both the epitheliumand the submucosa leading to the destruction of mucosal epithelium,which results in ulcerations, mainly in the mucous membranes lining theoral and digestive tract. Mucositis results in severe pain, reducedquality of life, prolonged hospitalisation, increase risk of local andsystemic infection; this is an even more serious consequence ofmucositis, since the lesions can act as sites of secondary infectionsand as portals of entry for endogenous oral microorganisms. Thereforemucositis is a significant risk factor for life-threatening systemicinfection (which can be exacerbated by the concomitant neutropenia;another side effect associated with chemotherapy) and often compromisesour ability to treat the underlying cancer by delaying or truncatinganticancer therapy and/or impeding recovery. High-dose chemotherapy andradiation therapy selectively affect rapidly-dividing cells, bothcancerous and non-cancerous. Both normal mucosal cells and malignantcells share the characteristic of fast growing or cycling; the rapidcellular turnover displayed by mucosal lining is also common to othernormal tissues such as blood cells, hair and skin that are also affectedby anti-cancer therapies. Accordingly, chemotherapy and radiationtherapy that are directed to interrupting cancer cell growth are alsoaffecting fast proliferating cells in the body, such as the mucosallining. This widely accepted explanation points out why mucositis oftenarises as a moderate to severe complication of antineoplastic therapysuch as cancer chemotherapy and/or radiation therapy (M. Duncan, GrantG., Aliment. Pharmacol. Ther., 18, 9, 853-74, 2003).

The best described mucositis are the ones which occur in the mouth (oralmucositis, OM) and in the gastrointestinal (GI) tract (GI mucositis,GIM), OM is a painful condition that significantly impairs chewing andswallowing, while GIM is becoming increasingly recognized as a toxicityassociated with many standard-dose chemotherapy regimen commonly used inthe treatment of cancer (chemotherapy-induced mucositis is present in40-100% of patients) and with radiotherapy addressed to any part of GItract. The small intestine is the most concerned, but also oesophagus,stomach and large intestine can be affected.

As the mucosa of the oral cavity and of the gastrointestinal tract sharea common embryological origin and development it is likely they sharethe same basic pathogenesis with only some differences due to specificfunctional components of intestinal tract. The damage to the intestineis similar to the damage that occurs in oral mucosa but it acts at amuch faster rate. Similar to OM, GIM is not solely due to a directcytotoxicity effect of radiotherapy but to a sum of direct (clonogenicand apoptotic cell death) and indirect (reactive changes) effects.

The acute toxicity in GIM could be accounted for the large part to cryptcell death, resulting in the breakdown of the mucosal barrier (Sonis S Tet al., Cancer, suppl. 100, 9, 1995-2025, 2004). This pivotal effect maybe the result of an effect either direct or mediated by a series ofintermediate steps as the crypt cell death could be a consequence ofendothelial apoptosis that, as in oral mucositis, become the primaryevent. As previously reported, many chemotherapeutic agents kill rapidlydividing cells, making GI tract particularly vulnerable, but differentlyfrom radiation, chemotherapy-induced mucositis have been focused mainlyon the small intestine. Cytotoxic agents act at different levels of thecrypt cell hierarchy, leading to crypt hypoplasia followed byregeneration. The first abnormality noted in human small intestine is anincrease in apoptosis on day 1 after chemotherapy; this is followed byreductions in crypt length, villus area, and mitotic index, which reachtheir maximal reduction on day 3. Rebound hyperplasia follows on day 5,prior to normalisation. Although more molecular events have beenelucidated in the pathogenesis of oral mucositis relative to its GIcounterpart, oral cavity and the GI tract have sufficient homology toexpect that mucosal barrier injury in the GI tract and in the oralmucosa share similar mechanisms (Sonis S T et al, Cancer, suppl. 100, 9,1995-2025, 2004).

Even though mucositis represents a clinical outcome due to a complexinteraction of local tissue (connective tissue, endothelium, epithelium)toxicity, induced by chemotherapy or radiation and could be seen asdifferent pathologies, recent scientific efforts in this areahighlighted how a common mechanistic scheme could be recognized for thephysiological basis of mucositis.

As a matter of fact, the evolution of mucosal barrier injury can beviewed as a five-phase process: the initial phase (step 1) ischaracterized by the generation of Reactive Oxygen Species (ROS). Thisis supported by studies reporting an attenuation of mucosal injuryinduced by agents that block or scavenge oxygen-free radicals (Facorro Get al., Bone Marrow Transplant., 33, 8, 793-8, 2004, Sonis S T et al.,Cancer, suppl. 100, 9, 1995-2025, 2004). The second phase (step 2) ischaracterized by a series of multiple effects driven from oxidativestress. Even though ROS can directly damage DNA (leading thus to thesubsequent clonogenic cell death), the more striking effect mediated byROS is the amplification of the damage, by stimulating a number oftranscription factors (Sonis S T et al., Cell Prolif., 35, Suppl1:93-102, 2002). Among them, nuclear factor-kB (NF-kB) has beenhighlighted as the key element in the genesis of mucositis (Sonis S T,Nat rev Cancer, 4, 4, 277-284, 2004). NF-kB is either activated bychemotherapy or radiotherapy and it is able to up-regulate a large panelof genes, including those that result in the production ofpro-inflammatory cytokines TNF_(α), IL-1 and IL-6, all leading toapoptosis and tissue injury, and up-regulation of genes that can causethe expression of adhesion molecules, cyclooxygnase-2 and iNOS. Theeffect of COX-2 and iNOS products in amplifying the tissue degenerationin experimental radiation-induced mucositis has been recently describedin depth (Sonis S T et al., Oral Oncol., 40, 2, 170-6, 2004; The thirdphase (step 3) is characterized by the amplification of signalingtriggered by pro-inflammatory cytokines that can activate differentpathways such as ceramide and caspase pathways, all leading to a furtherincrease in pro-inflammatory cytokines. The fourth step (step 4) ischaracterized by the symptoms of mucosal barrier destruction due totissue ulceration. During this phase there is a massive infiltration ofinflammatory cells and colonisation sustained by gram-positive andgram-negative bacteria. The cell wall products from bacteria can in turnactivate cell tissue infiltrate and exacerbate the inflammatoryreaction. This phase is very crucial for the continuation of cancertherapy and represents a serious risk of bacteraemia and/or fungalinfections.

The final phase (step 5), which occurs only in the absence ofinfections, represents the healing phase, that starts from extracellularmatrix and leads to renewal of epithelial proliferation anddifferentiation. After the healing phase the oral mucosa appears normal:however the mucosal environment has been altered and the patients is atrisk of future episode of mucositis during anticancer therapy.

This complex biological scenario, which shows how mucositis should beconsidered as the result of cumulative and interactive effects ofchemotherapy and/or radiation with epithelial connective tissue,endothelium, pro-inflammatory cytokines, cellular elements within themucosa as well as concomitant infections, may explain why the treatmentof mucositis has been so largely empirical and, due to the lack of aspecific and effective treatment, lead to either cessation of theanticancer therapy or consist of palliative and supportive intervention(Rubenstein E B, et al., Cancer suppl., 100, 9, 2026-2046, 2004;Worthington H V et al. Cochrane Review, 3, 2004). WO 99/45910 describesa method of treating mucositis by a mixture of therapeutic agents, suchas an NSAID, a MMP inhibitor, a NO inhibitor, a mast cell inhibitor andan inflammatory cytokine inhibitor. However, there is no experimentalevidence of therapeutic effectiveness of these mixtures. For OM it iswidely accepted that a good oral hygiene reduces the risk. Oral careprotocols are widely used with the purpose of maintaining mucosal healthand integrity, to reduce the impact of the oral microbial flora and toreduce symptoms such as pain and bleeding and prevent soft tissueinfections that may have systemic effects. In patients undergoinghaematopoietic stem cell transplantation the treatment of choice forpain control is the analgesia with morphine. Other approaches includethe use of systemic analgesics and palliative mixture of agents, coatingagent and topical analgesics. There is no significant evidence of theeffectiveness of this mixture. In patients with head and neck cancertreated with moderate radiotherapy the preventive pharmacologicalprotocol suggests topical use of benzydamine, due to itsanti-inflammatory effects beside to its analgesic and anaestheticproperties. Even though benzydamine has been extensively studied, thereare no definitive trials confirming its activity in preventing or cureradiation-induced mucositis. Also to treat chemotherapy-inducedmucositis: only palliative protocols are available. For high-dosechemotherapy the protocol recommends Low-Level Laser Therapy (LLLT) inan attempt to reduce the incidence of mucositis. It has been reportedthat LLLT promotes wound healing and reduces pain and inflammation.However this type of intervention requires a specific equipment, oftenexpensive, specialized training, and treatment can be time consuming.Finally, one drug is suggested to reduce esophagitis induced by combinedchemotherapy and radiotherapy, i.e. amifostine, due to its reportedradioprotective activity. Amifostine acts as a potent ROS scavenger,unfortunately this drug is endowed with a lot of negative features: itrequires iv administration, and it has acute toxicity. FDA approved itsclinical use only for reduction of renal toxicity associated withcisplatin therapy in patients bearing ovarian cancer or lung cancer.Only very recently the FDA has approved the use of the human recombinantkeratinocyte growth factor (rHu-KGF; palifermin), which, by increasingepithelial stem cell proliferation, differentiation and migrationensures an increase probability of epithelial cell survival and speed upthe rate of cell regeneration. Palifermin use is however restricted tothe treatment of mucositis only in adult patients with haematologicmalignancies undergoing myelotoxic therapy requiring haematopoietic stemcell transplant (the safety and efficacy of palifermin in the treatmentof mucositis has not been established in adult patients withnon-haematologic malignancies nor in children with both haematologic ornon-haematologic malignancies).

Due to the lack of effective pharmacological treatments, mucositisincidence is quite high in patients undergoing chemotherapy and/orradiation therapy or total body irradiation (the latter being theroutine preconditioning procedure prior to bone marrow transplant).

The incidence of oral and GI mucositis varied among therapy regimens(Sonis S T et al, Cancer, suppl. 100, 9, 1995-2025, 2004):anthracycline-based regimens were associated with 1-10%, as well as forpatients bearing breast cancer or non-Hodgkin lymphomas whose regimensnot included 5-FU. In contrast, chemotherapy including 5-FU wasassociated with more than 15% oral mucositis and chemotherapy withCPT-11 was associated with the same rate GI mucositis. Addition ofradiation to chemotherapy increased the risk to more than 30%. Oral andGI mucositis frequency and severity in patients undergoing high-dosechemotherapy combined with total body irradiation with haematopoieticstem cell transplantation can occur up to 100% of these patients and itis characterised by pain, difficulty to swallow to a total parenteralnutrition requirement, fever, risk of infection even to fatal sepsis.Radiation therapy to head and neck was associated with an even increasedincidence of oral or GI mucositis, often exceeding 50% of patients. Highfrequency and severity of mucositis is also present in patients with GIor gynaecologic malignancies. Acute damage to the GI mucosa is aconsequence of radiotherapy in 85-100% of patients.

This significant incidence of mucositis in patients undergoing cancertreatment also reflects in a relevant social cost, which includesincreased health care resource utilization, due to the reduction in curerate as a result of the reduction of the dose of the anticancer therapy,prolongation of hospitalisation for fever, narcotic usage and parenteralnutrition.

Finally, even though in a less extent, mucositis is not restricted tocancer patients only, since this disease also affects HIV patients,patients affected with non-Hodgkin's lymphoma, debilitated elderlypatients.

Accordingly, there is still a remarkable need for new therapieseffective in the treatment and prevention of mucositis.

GENERAL DESCRIPTION OF THE INVENTION

The subject matter of the inventions is defined by the appended claims.

The present invention relates to the use of a compound of formula (I)for preparing a medicament or pharmaceutical compositions containing aneffective amount of said compound for treatment and/or prevention ofmucositis.

Compounds of formula (I) represents a selected group of the compoundspreviously reported in the International Patent Application WO02/070468,from our group, and claimed for treatment of inflammatory andauto-immune diseases.

The present invention concerns the discovery that a selected group ofbenzamidine derivatives, those of formula (I) as reported above, areparticularly suitable for treatment and/or prevention of mucositis,particularly mucositis induced by chemotherapy and/or radiotherapy.

As detailed below, compounds of formula (I) are able to effectivelyinterfere with each of the mucositis phases, as described in thebackground, thus providing a highly efficacious pharmacological tool forprevention and treatment of mucositis.

More in detail, as reported in the background, mucositis (either OM orGIM) share a common degenerative pathway which involves five-phases orsteps. The first step is represented by the action of ROS that trigger acomplex series of events that characterize the second step, where, inaddition to the clonogenic cell death, the activation of nuclear factors(in particular NF-kB) leads to cytokines production along with otherpro-inflammatory agents (among them PGE₂ the main product of COX-2).During the third step the signal triggered by cytokines is amplified,giving rise to damage propagation which ultimately leads to tissueulceration. In the forth step mucosal barrier destruction occurs, andduring this phase there is a massive bacterial colonisation andinfiltration of inflammatory cells; finally during the fifth step, inthe absence of infection, healing occurs.

Compounds of formula (I) display a remarkable effect in preventing ROSformation in human cells, thus acting at Step 1 by preventing thetriggering process for mucositis. In addition the effect of compounds ofthis invention stretches to step 2, as highlighted by the potentinhibitory effect on cytokines production along with otherpro-inflammatory endogenous compounds such as prostaglandins (PGE₂) andnitric oxide (NO). In addition, compounds of formula (I) have been foundto be strongly effective in reducing the clonogenic stem cell death.Accordingly, acting at both the initiation step and the subsequentpropagation step, compounds of formula (I) are suitable agents for bothprevention and treatment of mucositis. Avoiding or reducing both theinsult and the subsequent propagation of the pro-inflammatory stimulus,these compounds exert they activity also in step 4, by preventing andtreating the concurrent damage to the basal epithelial cell and theconsequent mucosal breakdown, which is crucial for bacterialcolonisation, they can also indirectly prevent the infection. Finally,compounds of formula (I) have shown striking mucosal protective andwound-healing properties, thus these compounds are also able to actduring the healing phase (step 5).

Accordingly, this invention concerns with a new pharmacological therapyfor preventing and treating mucositis, which consists in administeringto a human in need thereof a pharmaceutical acceptable formulation of aneffective amount of a compound of formula (I) or its pharmaceuticallyacceptable salt or a solvate thereof.

The term “preventing” herein means any prophylactic action aimed atavoiding, inhibiting or restraining development of mucositis in apatient in need of this. The term “treating” includes prohibiting thedisease development, stopping or reversing its progression, decreasingseverity or resultant clinical symptoms of the disease, as well as anyimprovement in the well being of patients.

The term “mucositis” has the same meaning as described in thebackground, and refers to oral mucositis, gastrointestinal mucositis,uro-genital and nasal tract mucositis.

The patient treated with pharmaceutical compositions of the compounds ofthe invention can be a cancer patient preparing to undergo chemotherapyor radiation therapy, or a cancer patient currently undergoingchemotherapy or radiation therapy, or a patient preparing to bone marrowtransplant. In addition, HIV patients, patients affected withnon-Hodgkin's lymphoma, debilitated elderly patients, at risk orsuffering of mucositis can be treated with methods and compositions ofthis invention.

DETAILED DESCRIPTION OF THE INVENTION

Compounds of Formula (I):

Wherein:

-   -   A is selected independently from the thiocarboxamide and the        carboxamide groups.    -   R₁ is selected from an alkyl group having from 1 to 3 carbon        atoms and the amino group, unsubstituted or substituted with the        nitro group or the methyl group.    -   R₂ is selected independently from hydrogen, an alkyl group        having from 1 to 4 carbon atoms, a cycloalkane residue having        from 5 to 7 carbon atoms, an aryl, naphtyl or heterocyclic        group, unsubstituted or substituted with methyl, methoxy,        hydroxy, amino or halogen groups.    -   R₃ and R₄ are selected independently from hydrogen and an alkyl        group having from 1 to 3 carbon atoms.    -   R₅ represents one or two substituents independently selected        from hydrogen and the methyl, methoxy and hydroxyl groups,    -   n is a whole number from 0 to 6, and    -   the amidine group is in the para or meta position relative to        the “A-NH” group.

In the compounds of the invention, R₂ is linked to A through an alkylenegroup, having from 1 to 6 carbon atoms, optionally substituted with oneor more alkyl groups having from 1 to 3 carbon atoms.

In the compound of formula (I) an aryl group is a substituted or notsubstituted phenyl; an heterocyclic group is a monocyclic or bicyclicaromatic heterocycle containing 1 or 2 nitrogen atoms, or a monocyclicor bicyclic aromatic heterocycle containing 1 oxygen or sulphur atom.

Non limiting examples of heterocyclic groups are pyridine, furane,thiophene, quinoline benzofurane and benzothiophene.

The compounds of formula (I) used in the present invention can beprepared according to established procedures as described inWO02/070468, these procedures are summarized by reference herein. Ingeneral the process starts with the reaction of the appropriatelysubstituted phenylenediamine of formula (IV) (Scheme 1) which is reactedwith the corresponding isothiocyanate or isocyanate of formula (V a) or(V b) to give rise respectively to the corresponding thiourea of formula(III a) or urea of formula (III b). Compounds of formula (III) are thenreacted with the appropriate imidate hydrochloride of formula (II) toafford compounds of formula (I).

Scheme 1

Non limiting representative examples of compounds of formula (I) of thepresent invention are reported below and in Table 1:

-   -   N-[4-(N-acetamidine)phenyl]-N′-pentyl thiourea (compound 1.1)    -   1-guanidinophenyl-4-cyclohexyl thiourea (compound 1.2)    -   1-nitroguanidinophenyl-4-cyclohexyl thiourea (compound 1.3)    -   N-[4-(N-acetamidine)phenyl]-N′-butyl thiourea (compound 1.4)    -   N-[4-(N-acetamidine)phenyl]-N′-(3-methyl-butyl)thiourea        (compound 1.5)    -   N-[4-(N-acetamidine)phenyl]-N′-[2-(4-fluorophenyl)ethyl)thiourea        (compound 1.6)    -   N-[4-(N-acetamidine)phenyl]-N′-[2-(4-chlorophenyl)ethyl)thiourea        (compound 1.7)    -   N-[4-(N-acetamidine)phenyl]-N′-cyclohexyl urea (compound 1.8)

TABLE 1

Com- pound R₁ R₂ R₃/R₄ n A 1.1 CH₃ CH₃ H 4 NH—CS 1.2 NH₂ cyclohexyl — 0NH—CS 1.3 NO₂—NH cyclohexyl — 0 NH—CS 1.4 CH₃ CH₃ H 3 NH—CS 1.5 CH₃isopropyl H 2 NH—CS 1.6 CH₃ 4-F-Phenyl H 2 NH—CS 1.7 CH₃ 4-Cl-Phenyl H 2NH—CS 1.8 CH₃ cyclohexyl — 0 NH—CO

R₅ is always H in these compounds; the two phenyl N—H substituents arealways in the para position.

Pharmaceutically acceptable salts of compounds of formula (I) can beparticularly suitable for the preparation of pharmaceutical compositionsuseful for mucositis treatment, since they have enhanced watersolubility compared to the compound from which they are derived. Asreported below, for mucositis treatment and/or prevention in addition tothe usual oral formulations such as tablets, capsules and pills, alsosyrups, oral rinse, gels or emulsions can be useful formulations forthis disease treatment. In addition, a considerable water solubility isessential for a proper formulation of dosage forms for parenteraladministration, suitable for the treatment of the most severe forms ofthis disease. Finally, improved water solubility can also improveadsorption of oral formulations.

Salts of compound of formula (I) are typically formed by reacting acompound of formula (I) with an equimolar or excess amount of theappropriate acid.

Representative non limiting examples of pharmaceutically acceptablesalts of compounds of formula (I) are: hydrochloride, hydrobromide,hydrogensulphate and sulphate, methansulphonate, maleate, fumarate andsuccinate.

In order to provide examples about the impact on solubility exerted bydifferent pharmaceutically acceptable salts of compound of formula (I),the preparation of the maleate and of the methanesulphonate of compound1.1 is herein reported as non limiting representative example.

N-[4-(N-acetamidine)phenyl]-N′-pentyl thiourea maleate

Compound 1.1, 1 g (3.59 mmoles), is suspended in ethyl acetate (30 mL)then a solution of maleic acid, 416 mg (3.59 mmoles) in methanol (10 mL)is added on stirring at room temperature. The resulting solution isstirred at room temperature 10 minutes then concentrated in vacuum, theresulting residue is treated with a mixture of ethyl acetate (10 mL) andisopropylether (10 mL), the resulting precipitate is filtered and driedto afford 1.1 g of the maleate.

m.p. 215° C.; IR: 1681, 1622, 1543, 1511.

¹HNMR (DMSO-d₆), ppm: 0.90 (t, 3H, J=6.2 Hz); 1.31-1.36 (m, 4H);1.53-1.59 (m, 2H); 2.32 (s, 3H); 3.37-3.48 (m, 2H); 6.06 (s, 2H); 7.25(d, 2H, J=8.2); 7.69 (d, 2H, J=8.2 Hz); 7.94 (m, 1H); 8.48 (bs, 1H);9.43 (bs, 1H); 9.73 (bs, 1H);11.1 (s, 1H); 14.3 (s, 1H).

N-[4-(N-acetamidine)phenyl]-N′-pentyl thiourea methanesulphonate

This salt is prepared from 1 g of compound 1.1 and 0.23 mL (3.59 mmol)of methanesulphonic acid using the same procedure as reported above forthe maleate.

IR: 1676, 1627, 1544, 1511.

¹HNMR (DMSO-d₆), ppm: 0.93 (t, 3H, J=6.0 Hz); 1.30-1.38 (m, 4H);1.51-1.59 (m, 2H); 2.30 (s, 3H); 2.39 (s, 3H); 3.40-3.48 (m, 2H); 7.23(d, 2H, J=8.9); 7.69 (d, 2H, J=8.9 Hz); 8.04 (m, 1H); 8.50 (bs, 1H);9.43 (bs, 1H); 9.79 (bs, 1H);11.05 (s, 1H).

The hydrochloride of compound 1.1 is prepared as reported inWO02/070468.

Solubility in water, at 25° C., for the representative examples of saltsof compound 1.1 are reported in the table below:

Salts for Compound Solubility in water Solubility in water 1.1* (mg/mL)(%) Hydrochloride 9.0 0.90 Maleate 3.12 0.31 Methanesulphonate >32 >32*The compound 1.1 is not water soluble as free base.

Pharmacological Activity

The compounds of the invention have been demonstrated to inhibit ROSproduction in human polymorphonuclear leukocyte (PMNL), to inhibitcytokine production, iNOS and COX-2 protein expression, as assessed inan “in vitro” rat model, to protect the mucosa and display wound-healingproperties, as assessed in a rat model of gastric mucosa ulcerationinduced by indomethacin. Finally, the compounds of the inventionincrease crypt cell survival, as evaluated in an “in vivo” mucositismodel in mice.

As a representative non-limiting example pharmacological data forcompound 1.1 are reported below.

Inhibition of ROS Generation in Human PMNL:

Background to the assay: One of the most important events involved inthe intracellular cascade leading to NF-kB activation is the generationof oxidative stress and the increase of ROS; the inhibition of thesespecies can contribute to reduce the direct damage to DNA and subsequentclonogenic cell death, and also to diminish the transcription factorsactivation. The effect of compound 1.1 on luminol-dependentchemiluminescence assay was assessed in human PMNL. Data are reported inFIG. 1.

Cytokine Inhibition in Rat Peritoneal Macrophages.

Background to the assay: Nuclear factor-κB (NF-κB), a key element in thegenesis of mucositis, has the capacity to upregulate a large panel ofgenes, including those that result in the production of pro-inflammatorycytokines, TNF_(α), IL-1 and IL-6, all leading to apoptosis and tissueinjury, and to up-regulate genes that can cause the expression of iNOSand cyclooxygnase-2. The third phase of mucositis is indeedcharacterized by the amplification of signaling triggered bypro-inflammatory cytokines. The effect of compound 1.1 was assessed inrat peritoneal macrophages. Data are reported in table 1 and 2.

TABLE 1 Inhibition by compound 1.1 of LPS-induced cytokine release inrat peritoneal macrophages IC₅₀ (μM) Example IL-1β IL-6 TNFα Compound1.1 30 μM 63 μM 54 μM

TABLE 2 Inhibition by compound 1.1 of LPS-induced iNOs and COX-2 proteinexpression in rat macrophages % of inhibition treatment iNOs COX-2 LPS,0.1 μg/ml, 24 h 0 0 LPS, 0.1 μg/ml + Compound 79 53 1.1, 30 μM

Gastric Ulcer Induced By Indomethacin in Rat: Wound-Healing Propertiesof Compound 1.1

Indomethacin induces the formation of acute gastric mucosal lesions. Thehistologic damage is represented by necrosis with loss of surfaceepithelium, submucosal oedema and leukocyte infiltration. The mechanisminvolves a neutrophil-dependent process inducing a variety ofinflammatory mediators such as reactive oxygen species, and directdetrimental action by indomethacin on processes linked to epithelialproliferation and apoptosis. The epithelial repairing process is due tocontinuity of epithelial cells with healthy cells of gastric pits thatcan migrate to the basement membrane; re-epithelialization andreconstruction of the mucosal architecture is under the control ofgrowth factors produced locally by regenerating cells.

The effect of compound 1.1 was assessed in rat gastric mucosa. Data arereported in FIG. 2.

Finally, in vivo efficacy of the compounds of the invention wasdemonstrated in a mucositis model in mice.

Mouse Mucositis Model

There are thought to be between four and sixteen actual stem cells ineach crypt of the small intestine. There are also a further reserve ofclonogenic cells which are capable of regenerating the crypt when allthe actual stem cells have been killed. The survival of these clonogeniccells is therefore key to the survival of the crypt and the restorationof an intact epithelial lining following cytotoxic injury (only oneclonogenic cell needs to survive to ensure the survival of the crypt,and therefore the maintenance of an intact epithelium). Growth factorsand other molecules can be used to manipulate the sensitivity of thesecells to cytotoxic agents, and thereby reduce the severity ofgastrointestinal and oral mucositis. Factors given prior to a cytotoxicinsult may increase clonogenic cell number (thereby increasing theprobability of clonogen survival) or act to arrest the cell cycle insuch cells (thereby making them more resistant to damage or death).Factors given after the insult may initiate early stem cellamplification or proliferation and hence speed up the regenerationprocess. A combination of both protocols could give maximum protectionto the epithelium.

This study therefore examined the effectiveness of Compound 1.1 atprotecting clonogenic cells, and hence crypts, from radiation induceddamage. The effects of administration for 3 days before radiationexposure were tested.

The protective effect is summarized in FIG. 3 and detailed in table 3.

Compound 1.1 at 20 mg/kg prevented the absence of surviving crypt (asseen in 4% of circumference in vehicle treated mouse), and increased thepercentage of surviving crypts per circumference.

TABLE 3 Effect of Compound 1.1 on clonogenic crypt cell death induced bywhole body irradiation in mouse. corrected no. crypts/ crypt crypts/Treatment circumference width (μm) circumference 20 mg/kg Compound 1.1for 3 Average +/− sd 13.2 +/− 5.7  66.19 +/− 3.0 6.7 +/− 3.0 days pre -13Gy irradiation 10 mg/kg Compound 1.1 for 3 Average +/− sd 9.2 +/− 4.768.25 +/− 2.4 4.5 +/− 2.4 days pre - 13Gy irradiation 5 mg/kg Compound1.1 for 3 days Average +/− sd 9.6 +/− 3.4 68.54 +/− 6.6 4.7 +/− 1.3pre - 13Gy irradiation vehicle controls, 13Gy irradiation Average +/− sd7.2 +/− 3.9 68.92 +/− 6.1 3.5 +/− 1.9 untreated controls Average +/− sd102.8 +/− 5.8  33.66 +/− 1.3

Pharmacological Assays

Inhibition of Chemiluminescence in Human PMNL

Human neutrophils were obtained from healthy volunteers. Blood wasanti-coagulated with Na-Citrate 0.38% and neutrophils were purifiedaccording to Boyum (Boyum A. Scand J Clin Invest 1968;21:77-89). Theneutrophil purification was achieved by gradient centrifugation onHisto-paque at 400 g for 30 min. Resulting neutrophils were suspended inPBS plus 0.87 mM CaCl₂, 1 mM MgCl₂, counted, and diluted to 2.5×10⁶/ml.Neutrophil suspension was premixed with luminol (5 μM final). A 200μl-aliquot of cell suspension was incubated into a 96-well plate, withdrugs for 10 min at 37° C. Neutrophils were activated with 0.1 μMphorbol 12-myristate 13-acetate (PMA) and the light emission wasmonitored at 3 min intervals for 24 min in a HTS7000 plus microplatereader. Results were expressed as reduction of the fluorescent signalrecorded for cell activated with PMA alone.

Activation of neutrophils with 0.1 μM PMA induced a time-dependentincrease in signal luminescence, with a maximal increase within 10-12min. The pre-incubation with Compound 1.1 (1-10 μM)concentration-dependently decreased luminol-enhanced chemiluminescence(rising phase, at 15 min, IC₅₀=6.4±0.6 μM, steady phase, at 24 min,IC50=2.9±0.2 μM). The inhibitory effect was detectable even at thelowest concentration of 1 μM (20% of inhibition) and at 30 μM Compound1.1 completely inhibited ROS generation (data not shown). The data areillustrated in FIG. 1.

Cytokine Inhibition in Rat Peritoneal Macrophages

Primary cell cultures were obtained from male albino rats (SD, 200-250g, Harlan, Italy), as described in Methods in Enzymology (Methods inEnzymology, vol. LVIII, pages 494-506). Cells were stimulated, the dayafter plating, with LPS, 1 μg/ml or 0.1 μg/ml as stated, for 24 h.Compounds were added 20 min before stimulation. The stimulation wasperformed in DMEM, 1 g/l glucose, 50 μg/ml gentamicin. Supernatants andcell lysates were collected and stored at −80° C. until use. Cytokinequantisation in supernatants was determined by means of commerciallyavailable ELISA Kits for rat TNFα, rat IL-1β and rat IL-6 (Amersham).

Western blot analysis of iNOS and COX-2: Cell lysates were analyzed bySDS-PAGE. Proteins were transferred onto PVDF membranes and saturated inblocking buffer. Membranes were incubated for 2 h at RT with thefollowing antibodies: anti-COX-2, anti-iNOS, anti-β-actin and furtherincubated with a secondary antibody for 45 min at RT. Detection wasperformed using ECL (Amersham). Quantisation was determined bydensitometry analysis using NIH Image software.

Activation of macrophages with 1 μg/ml LPS induced an increase overbasal in cytokine production. The pre-incubation with Compound 1.1(3-100 μM) concentration-dependently decreased all the three cytokines,i.e. IL-1β, IL-6 and TNFα. Compound 1.1 in the range 30-100 μMsignificantly inhibited cytokine production The data are illustrated intable 1.

Evaluation of mediator of inflammation such as iNOS and COX-2, wasperformed in macrophages stimulated with 0.1 μg/ml LPS for 24 h.Compound 1.1, tested at 30 μM, significantly decrease both iNOs andCOX-2 protein expression. The data are illustrated in table 2.

Gastric Ulcer Induced By Indomethacin in Rat: Wound-Healing Propertiesof Compound 1.1

20 male SD rats (140-160) were used. The animals were deprived of foodbut not water 24 hours prior the experiment. Gastric ulcer was inducedin conscious rats by oral administration of 10 mg/kg/4 ml ofindomethacin, suspended in methylcellulose 0.5%. The tested drug wasadministered 30 min by gavage (os), or 15 min by subcutaneously (sc),before indomethacin.

Four hours after indomethacin administration, the animals weresacrificed by excess of ether. The stomach was dissected out, openedalong the greater curvature, and the mucosa was examined by an observerwho was unaware of the treatment given. The extent of ulcers wasmeasured with a 10× binocular fitted with a 0.1 mm-division scale. Dataare presented as total length of ulcers per group.

The animals were divided into 4 groups of 5 animals, and were treated asfollows:

Groups:

-   -   1. 10 mg/kg Compound 1.1, administered prior to 10 mg/kg        indomethacin, os.    -   2. 5 mg/kg Compound 1.1, administered prior to 10 mg/kg        indomethacin, os.    -   3. 1 mg/kg Compound 1.1, administered prior to 10 mg/kg        indomethacin, os.    -   4. vehicle, administered prior to 10 mg/kg indomethacin, os.

Compound 1.1 was given at 1, 5 and 10 mg/kg prior to indomethacin. Invehicle treated groups all the animals exhibited ulcers. In Compound 1.1treated groups the levels of ulcerated animals decreaseddose-dependently. Maximal effect, i.e. no animals ulcerated, wasachieved at the highest dose (10 mg/kg). The 5 mg/kg dose reduced aboutto 50% the incidence of ulcer in both administration protocol (3/5animals), and, most important, the extent of ulceration was dramaticallyreduced up to 80-90%. The lower dose (1 mg/kg) was effective only in theos protocol administration.

All the animals survived the treatment and exhibited no obvious adverseeffects.

The data are illustrated in FIG. 2.

Mouse Mucositis Model

30 adult male BDF1 mice (aged 10-12 weeks) were used. The animals werehoused for 2 weeks in individually ventilated cages on a 12 hrlight:dark cycle to stabilize the circadian rhythm. Animals were allowedfood and water ad libitum throughout.

The animals were divided into 5 groups of 6 animals, and were treated asfollows:

Groups:

-   -   1. Gavage 20 mg/kg Compound 1.1 72, 48 and 24 hrs prior to 13Gy        X-ray exposure (whole body).    -   2. Gavage 10 mg/kg Compound 1.1 72, 48 and 24 hrs prior to 13Gy        X-ray exposure (whole body).    -   3. Gavage 5 mg/kg Compound 1.1 72, 48 and 24 hrs prior to 13Gy        X-ray exposure (whole body).    -   4. Gavage vehicle 72, 48 and 24 hrs prior to 13Gy X-ray exposure        (whole body).    -   5. Untreated, un-irradiated controls.

Intestinal damage was induced using a single dose of 13Gy X-irradiation.Four days after irradiation the animals were sacrificed. The smallintestine was removed and fixed in Carnoy's fixative prior to processingfor histological analysis. 3 μm sections were cut and stained withhaematoxylin and eosin. Foci of regeneration (surviving crypts with oneor more clonogenic cells) were clearly visible in the irradiatedsections. Other than these foci the mesenchyme was entirely denuded;these animals would develop diarrhea and die due to mucositis if allowedto live beyond four days.

For each animal ten intestinal circumferences were analyzed (60 pergroup)—a circumference is equivalent to a given length of intestine andtherefore a convenient baseline unit of length. The number of survivingcrypts per circumference was scored and the average per groupdetermined. Only crypts containing 10 or more strongly haematoxylin andeosin stained cells (excluding Paneth cells) and only intactcircumferences not containing Peyers patches were scored (Peyers patchesinfluence both the number of crypts in a normal circumference and theability of a crypt to survive insult).

The average crypt width (measured at its widest point) was also measuredin order to correct for scoring errors due to crypt size difference. Thecorrection is applied thus: Corrected number of crypts/circumference=

${{Corrected}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{14mu} {{crypts}/{circumference}}} = {\frac{{Mean}\mspace{14mu} {crypt}\mspace{14mu} {width}\mspace{14mu} {in}\mspace{14mu} {untreated}\mspace{14mu} {control}}{{Mean}\mspace{14mu} {crypt}\mspace{14mu} {width}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {animal}} \times {Mean}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{14mu} {surving}\mspace{14mu} {crypts}\mspace{14mu} {in}\mspace{14mu} {treatment}\mspace{14mu} {group}}$

Compound 1.1 was given at 5, 10 and 20 mg/kg daily for three days priorto radiation exposure. In animals treated with the vehicle 3.5±1.9crypts per circumference (cross section) survived the insult. In eachCompound 1.1 treated group the levels of survival were increased.Maximal survival was achieved at the highest dose (20 mg/kg) where6.7±3.0 crypts survived (1.9× increase). The lower doses increasedsurvival about 1.3 times. These levels of protection can allow animalsurvival following an otherwise lethal dose of irradiation (assumingbone marrow damage is minimized) (Both reviewed in Booth & Potten 2001,JNCI Monogr, 29; 16-20).

All the animals survived the treatment and exhibited no obvious adverseeffects.

The data are illustrated in Table 3 and FIG. 3.

Pharmaceutical Compositions

The route of administration is governed by the physical properties ofthe compound used and the type of mucositis to be treated and/orprevented. As discussed above, for mucositis treatment and/or preventionthe compounds of formula (I) can be administered as oral formulationssuch as tablets, capsules, pills, or as syrups, oral rinse, gels andemulsions. Since the composition of the invention can be used also forpreventing mucositis, administration of the compositions shouldpreferably precede the initial dose of antineoplastic therapy or theradiation therapy by at least 24 hours.

The particular dosage of compounds of formula (I) required to prevent ortreat mucositis or its symptoms, according to this invention, willdepend upon the severity of the condition, the route of administrationand the related factors that will be decided by the attending physician.Generally, accepted and effective oral daily doses will be from about0.5 to 500 mg/day (and more typically from about 10 to 100 mg/day). Suchdosages will be administered to a subject in need thereof from once toabout three times each day, or more often as needed, and for asufficient duration, to effectively inhibit mucositis.

Suitable pharmaceutical compositions of compounds of formula (I) can beprepared by procedures known in the art. For example the compounds canbe formulated with common excipients, diluents or carriers and formedinto tablets, capsules, pills, mouth washes, suspensions or gels.

Examples of excipients, diluents, and carriers that are suitable forsuch formulations include but not limit to: fillers and extenders suchas starch, lactose, mannitol, and silica derivatives; binding agentssuch as carboxymethyl cellulose and other cellulose derivatives,alginates, polyvinyl pyrrolidone.

Disintegrating agents such as calcium carbonate or sodium bicarbonatecan be added where required. Lubricants such as talc, calcium andmagnesium stearate or solid polyethyl glycols can be used for thesecompositions manufacturing, depending upon the physical properties ofthe compound of formula (I) to be formulated.

The compounds of the invention can also be formulated as suspensions orsolutions for convenient oral administration or as solutions appropriatefor parenteral administration, for instance by intramuscular,subcutaneous, or intravenous routes. The compositions of the inventioncan be in the form of a slightly viscous aqueous liquid (gel), whichprovides a film-forming and coating effect on the epithelial surfacessuch as, but not limited to the oral mucosa.

1. The use of a compound of formula (I), or its pharmaceuticallyacceptable salt or solvate, for preparing a medicament for treating orpreventing mucositis induced by cancer therapy comprising chemotherapyand/or radiotherapy:

wherein: A is selected independently from the thiocarboxamide and thecarboxamide groups, R₁ is selected from an alkyl group having from 1 to3 carbon atoms and the amino group, unsubstituted or substituted withthe nitro group or the methyl group, R₂ is selected independently fromhydrogen, an alkyl group having from 1 to 4 carbon atoms, a cycloalkaneresidue having from 5 to 7 carbon atoms, an aryl, naphtyl orheterocyclic group, unsubstituted or substituted with methyl, methoxy,hydroxy, amino or halogen groups, R₃ and R₄ are selected independentlyfrom hydrogen and an alkyl group having from 1 to 3 carbon atoms, R₅represents one or two substituents independently selected from hydrogenand the methyl, methoxy and hydroxyl groups, n is a whole number from 0to 6, and the amidine group is in the para or meta position relative tothe “A-NH” group or a pharmaceutically acceptable salt or solvatethereof.
 2. The use according to claim 1, wherein in the compounds offormula (I) A is the thiocarboxamide group, R₁ and R₂ are methyl groups,R₃ and R₄ and R₅ are hydrogen, n is a whole number from 0 to 6, and theamidine group is in the para position relative to the “A-NH” group, or apharmaceutically acceptable salt or solvate thereof.
 3. The use ofaccording to claim 1, wherein in the compounds of formula (I) A is thethiocarboxamide group, R₁ is selected from the amino group,unsubstituted or substituted with the nitro or methyl group, R₂ ismethyl, R₃ and R₄ and R₅ are hydrogen, n is a whole number from 0 to 6,and the amidine group is in the para or meta position relative to the“A-NH” group, or a pharmaceutically acceptable salt or solvate thereof.4. The use according to claim 1, wherein in the compounds of formula (I)A is the thiocarboxamide group, R₁ is a methyl group, R₂ is an arylgroup, R₃ and R₄ and R₅ are hydrogen, n is a whole number from 0 to 6,and the amidine group is in the para position relative to the “A-NH”group, or a pharmaceutically acceptable salt or solvate thereof.
 5. Theuse according to claim 1, wherein in the compounds of formula (I) A isthe thiocarboxamide group, R₁ is a methyl group, R₂ is an heterocyclicgroup, R₃ and R₄ and R₅ are hydrogen, n is a whole number from 0 to 6,and the amidine group is in the para position relative to the “A-NH”group, or a pharmaceutically acceptable salt or solvate thereof.